5,574 research outputs found

    RNA pull-down-confocal nanoscanning (RP-CONA), a novel method for studying RNA/protein interactions in cell extracts that detected potential drugs for Parkinson’s disease targeting RNA/HuR complexes

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    MicroRNAs (miRNAs, miRs) are a class of small non-coding RNAs that regulate gene expression through specific base-pair targeting. The functional mature miRNAs usually undergo a two-step cleavage from primary miRNAs (pri-miRs), then precursor miRNAs (pre-miRs). The biogenesis of miRNAs is tightly controlled by different RNA-binding proteins (RBPs). The dysregulation of miRNAs is closely related to a plethora of diseases. Targeting miRNA biogenesis is becoming a promising therapeutic strategy. HuR and MSI2 are both RBPs. MiR-7 is post-transcriptionally inhibited by the HuR/MSI2 complex, through a direct interaction between HuR and the conserved terminal loop (CTL) of pri-miR-7-1. Small molecules dissociating pri-miR-7/HuR interaction may induce miR-7 production. Importantly, the miR-7 levels are negatively correlated with Parkinson’s disease (PD). PD is a common, incurable neurodegenerative disease causing serious motor deficits. A hallmark of PD is the presence of Lewy bodies in the human brain, which are inclusion bodies mainly composed of an aberrantly aggregated protein named α-synuclein (α-syn). Decreasing α-syn levels or preventing α-syn aggregation are under investigation as PD treatments. Notably, α-syn is negatively regulated by several miRNAs, including miR-7, miR-153, miR-133b and others. One hypothesis is that elevating these miRNA levels can inhibit α-syn expression and ameliorate PD pathologies. In this project, we identified miR-7 as the most effective α-syn inhibitor, among the miRNAs that are downregulated in PD, and with α-syn targeting potentials. We also observed potential post-transcriptional inhibition on miR-153 biogenesis in neuroblastoma, which may help to uncover novel therapeutic targets towards PD. To identify miR-7 inducers that benefit PD treatment by repressing α-syn expression, we developed a novel technique RNA Pull-down Confocal Nanoscaning (RP-CONA) to monitor the binding events between pri-miR-7 and HuR. By attaching FITC-pri-miR-7-1-CTL-biotin to streptavidin-coated agarose beads and incubating them in human cultured cell lysates containing overexpressed mCherry-HuR, the bound RNA and protein can be visualised as quantifiable fluorescent rings in corresponding channels in a confocal high-content image system. A pri-miR-7/HuR inhibitor can decrease the relative mCherry/FITC intensity ratio in RP-CONA. With this technique, we performed several small-scale screenings and identified that a bioflavonoid, quercetin can largely dissociate the pri-miR-7/HuR interaction. Further studies proved that quercetin was an effective miR-7 inducer as well as α-syn inhibitor in HeLa cells. To understand the mechanism of quercetin mediated α-syn inhibition, we tested the effects of quercetin treatment with miR-7-1 and HuR knockout HeLa cells. We found that HuR was essential in this pathway, while miR-7 hardly contributed to the α-syn inhibition. HuR can directly bind an AU-rich element (ARE) at the 3’ untranslated region (3’-UTR) of α-syn mRNA and promote translation. We believe quercetin mainly disrupts the ARE/HuR interaction and disables the HuR-induced α-syn expression. In conclusion, we developed and optimised RP-CONA, an on-bead, lysate-based technique detecting RNA/protein interactions, as well as identifying RNA/protein modulators. With RP-CONA, we found quercetin inducing miR-7 biogenesis, and inhibiting α-syn expression. With these beneficial effects, quercetin has great potential to be applied in the clinic of PD treatment. Finally, RP-CONA can be used in many other RNA/protein interactions studies

    Molecular Tools for the Study of Resistance to Disinfectants

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    Disinfectants, antiseptics, and sanitizers are crucial for hygiene standards and disease control, as recently emphasized by the SARS-CoV-2 (COVID-19) pandemic. With the foreshadowing of antibiotic resistance, new cutting-edge technologies and innovative methodology need to be applied to prevent the latest emerging antimicrobial resistance crisis, resistance to disinfectants. Disinfectant resistance is a relatively novel field of study, and although some molecular mechanisms have been elucidated, little is known about complex mechanisms, cross-resistance with antibiotics, and the existence of resistance biomarkers. Fortunately, great advances have been made in the field of sequencing technology and bioinformatics. Although there are many limitations to this technology, various “omics” approaches to disinfectant resistance will be crucial in directing environment-specific disinfection programs. In addition, the vast amounts of data generated by sequencing technologies can be applied by artificial intelligence (AI) models to identify key disinfectant resistance markers and in the surveillance of disinfectant resistance genes. A combination of these approaches will be crucial in identifying new disinfectant resistance mechanisms, in monitoring resistant populations, and in identifying cellular targets for new disinfectant formulations. These molecular tools will be vital in the battle against disinfectant resistance, the latest development in the antimicrobial resistance crisis

    SYSTEMS METHODS FOR ANALYSIS OF HETEROGENEOUS GLIOBLASTOMA DATASETS TOWARDS ELUCIDATION OF INTER-TUMOURAL RESISTANCE PATHWAYS AND NEW THERAPEUTIC TARGETS

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    In this PhD thesis is described an endeavour to compile litterature about Glioblastoma key molecular mechanisms into a directed network followin Disease Maps standards, analyse its topology and compare results with quantitative analysis of multi-omics datasets in order to investigate Glioblastoma resistance mechanisms. The work also integrated implementation of Data Management good practices and procedures

    Hunting Wildlife in the Tropics and Subtropics

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    The hunting of wild animals for their meat has been a crucial activity in the evolution of humans. It continues to be an essential source of food and a generator of income for millions of Indigenous and rural communities worldwide. Conservationists rightly fear that excessive hunting of many animal species will cause their demise, as has already happened throughout the Anthropocene. Many species of large mammals and birds have been decimated or annihilated due to overhunting by humans. If such pressures continue, many other species will meet the same fate. Equally, if the use of wildlife resources is to continue by those who depend on it, sustainable practices must be implemented. These communities need to remain or become custodians of the wildlife resources within their lands, for their own well-being as well as for biodiversity in general. This title is also available via Open Access on Cambridge Core

    Developing automated meta-research approaches in the preclinical Alzheimer's disease literature

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    Alzheimer’s disease is a devastating neurodegenerative disorder for which there is no cure. A crucial part of the drug development pipeline involves testing therapeutic interventions in animal disease models. However, promising findings in preclinical experiments have not translated into clinical trial success. Reproducibility has often been cited as a major issue affecting biomedical research, where experimental results in one laboratory cannot be replicated in another. By using meta-research (research on research) approaches such as systematic reviews, researchers aim to identify and summarise all available evidence relating to a specific research question. By conducting a meta-analysis, researchers can also combine the results from different experiments statistically to understand the overall effect of an intervention and to explore reasons for variations seen across different publications. Systematic reviews of the preclinical Alzheimer’s disease literature could inform decision making, encourage research improvement, and identify gaps in the literature to guide future research. However, due to the vast amount of potentially useful evidence from animal models of Alzheimer’s disease, it remains difficult to make sense of and utilise this data effectively. Systematic reviews are common practice within evidence based medicine, yet their application to preclinical research is often limited by the time and resources required. In this thesis, I develop, build-upon, and implement automated meta-research approaches to collect, curate, and evaluate the preclinical Alzheimer’s literature. I searched several biomedical databases to obtain all research relevant to Alzheimer’s disease. I developed a novel deduplication tool to automatically identify and remove duplicate publications identified across different databases with minimal human effort. I trained a crowd of reviewers to annotate a subset of the publications identified and used this data to train a machine learning algorithm to screen through the remaining publications for relevance. I developed text-mining tools to extract model, intervention, and treatment information from publications and I improved existing automated tools to extract reported measures to reduce the risk of bias. Using these tools, I created a categorised database of research in transgenic Alzheimer’s disease animal models and created a visual summary of this dataset on an interactive, openly accessible online platform. Using the techniques described, I also identified relevant publications within the categorised dataset to perform systematic reviews of two key outcomes of interest in transgenic Alzheimer’s disease models: (1) synaptic plasticity and transmission in hippocampal slices and (2) motor activity in the open field test. Over 400,000 publications were identified across biomedical research databases, with 230,203 unique publications. In a performance evaluation across different preclinical datasets, the automated deduplication tool I developed could identify over 97% of duplicate citations and a had an error rate similar to that of human performance. When evaluated on a test set of publications, the machine learning classifier trained to identify relevant research in transgenic models performed was highly sensitive (captured 96.5% of relevant publications) and excluded 87.8% of irrelevant publications. Tools to identify the model(s) and outcome measure(s) within the full-text of publications may reduce the burden on reviewers and were found to be more sensitive than searching only the title and abstract of citations. Automated tools to assess risk of bias reporting were highly sensitive and could have the potential to monitor research improvement over time. The final dataset of categorised Alzheimer’s disease research contained 22,375 publications which were then visualised in the interactive web application. Within the application, users can see how many publications report measures to reduce the risk of bias and how many have been classified as using each transgenic model, testing each intervention, and measuring each outcome. Users can also filter to obtain curated lists of relevant research, allowing them to perform systematic reviews at an accelerated pace with reduced effort required to search across databases, and a reduced number of publications to screen for relevance. Both systematic reviews and meta-analyses highlighted failures to report key methodological information within publications. Poor transparency of reporting limited the statistical power I had to understand the sources of between-study variation. However, some variables were found to explain a significant proportion of the heterogeneity. Transgenic animal model had a significant impact on results in both reviews. For certain open field test outcomes, wall colour of the open field arena and the reporting of measures to reduce the risk of bias were found to impact results. For in vitro electrophysiology experiments measuring synaptic plasticity, several electrophysiology parameters, including magnesium concentration of the recording solution, were found to explain a significant proportion of the heterogeneity. Automated meta-research approaches and curated web platforms summarising preclinical research could have the potential to accelerate the conduct of systematic reviews and maximise the potential of existing evidence to inform translation

    Principles of Massively Parallel Sequencing for Engineering and Characterizing Gene Delivery

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    The advent of massively parallel sequencing and synthesis technologies have ushered in a new paradigm of biology, where high throughput screening of billions of nucleid acid molecules and production of libraries of millions of genetic mutants are now routine in labs and clinics. During my Ph.D., I worked to develop data analysis and experimental methods that take advantage of the scale of this data, while making the minimal assumptions necessary for deriving value from their application. My Ph.D. work began with the development of software and principles for analyzing deep mutational scanning data of libraries of engineered AAV capsids. By looking at not only the top variant in a round of directed evolution, but instead a broad distribution of the variants and their phenotypes, we were able to identify AAV variants with enhanced ability to transduce specific cells in the brain after intravenous injection. I then shifted to better understand the phenotypic profile of these engineered variants. To that end, I turned to single-cell RNA sequencing to seek to identify, with high resolution, the delivery profile of these variants in all cell types present in the cortex of a mouse brain. I began by developing infrastructure and tools for dealing with the data analysis demands of these experiments. Then, by delivering an engineered variant to the animal, I was able to use the single-cell RNA sequencing profile, coupled with a sequencing readout of the delivered genetic cargo present in each cell type, to define the variant’s tropism across the full spectrum of cell types in a single step. To increase the throughput of this experimental paradigm, I then worked to develop a multiplexing strategy for delivering up to 7 engineered variants in a single animal, and obtain the same high resolution readout for each variant in a single experiment. Finally, to take a step towards translation to human diagnostics, I leveraged the tools I built for scaling single-cell RNA sequencing studies and worked to develop a protocol for obtaining single-cell immune profiles of low volumes of self-collected blood. This study enabled repeat sampling in a short period of time, and revealed an incredible richness in individual variability and time-of-day dependence of human immune gene expression. Together, my Ph.D. work provides strategies for employing massively parallel sequencing and synthesis for new biological applications, and builds towards a future paradigm where personalized, high-resolution sequencing might be coupled with modular, customized gene therapy delivery.</p

    Genome-wide identification of the genetic basis of amyotrophic lateral sclerosis

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    Amyotrophic lateral sclerosis (ALS) is a complex disease that leads to motor neuron death. Despite heritability estimates of 52%, genome-wide association studies (GWASs) have discovered relatively few loci. We developed a machine learning approach called RefMap, which integrates functional genomics with GWAS summary statistics for gene discovery. With transcriptomic and epigenetic profiling of motor neurons derived from induced pluripotent stem cells (iPSCs), RefMap identified 690 ALS-associated genes that represent a 5-fold increase in recovered heritability. Extensive conservation, transcriptome, network, and rare variant analyses demonstrated the functional significance of candidate genes in healthy and diseased motor neurons and brain tissues. Genetic convergence between common and rare variation highlighted KANK1 as a new ALS gene. Reproducing KANK1 patient mutations in human neurons led to neurotoxicity and demonstrated that TDP-43 mislocalization, a hallmark pathology of ALS, is downstream of axonal dysfunction. RefMap can be readily applied to other complex diseases

    Machine learning and large scale cancer omic data: decoding the biological mechanisms underpinning cancer

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    Many of the mechanisms underpinning cancer risk and tumorigenesis are still not fully understood. However, the next-generation sequencing revolution and the rapid advances in big data analytics allow us to study cells and complex phenotypes at unprecedented depth and breadth. While experimental and clinical data are still fundamental to validate findings and confirm hypotheses, computational biology is key for the analysis of system- and population-level data for detection of hidden patterns and the generation of testable hypotheses. In this work, I tackle two main questions regarding cancer risk and tumorigenesis that require novel computational methods for the analysis of system-level omic data. First, I focused on how frequent, low-penetrance inherited variants modulate cancer risk in the broader population. Genome-Wide Association Studies (GWAS) have shown that Single Nucleotide Polymorphisms (SNP) contribute to cancer risk with multiple subtle effects, but they are still failing to give further insight into their synergistic effects. I developed a novel hierarchical Bayesian regression model, BAGHERA, to estimate heritability at the gene-level from GWAS summary statistics. I then used BAGHERA to analyse data from 38 malignancies in the UK Biobank. I showed that genes with high heritable risk are involved in key processes associated with cancer and are often localised in genes that are somatically mutated drivers. Heritability, like many other omics analysis methods, study the effects of DNA variants on single genes in isolation. However, we know that most biological processes require the interplay of multiple genes and we often lack a broad perspective on them. For the second part of this thesis, I then worked on the integration of Protein-Protein Interaction (PPI) graphs and omics data, which bridges this gap and recapitulates these interactions at a system level. First, I developed a modular and scalable Python package, PyGNA, that enables robust statistical testing of genesets' topological properties. PyGNA complements the literature with a tool that can be routinely introduced in bioinformatics automated pipelines. With PyGNA I processed multiple genesets obtained from genomics and transcriptomics data. However, topological properties alone have proven to be insufficient to fully characterise complex phenotypes. Therefore, I focused on a model that allows to combine topological and functional data to detect multiple communities associated with a phenotype. Detecting cancer-specific submodules is still an open problem, but it has the potential to elucidate mechanisms detectable only by integrating multi-omics data. Building on the recent advances in Graph Neural Networks (GNN), I present a supervised geometric deep learning model that combines GNNs and Stochastic Block Models (SBM). The model is able to learn multiple graph-aware representations, as multiple joint SBMs, of the attributed network, accounting for nodes participating in multiple processes. The simultaneous estimation of structure and function provides an interpretable picture of how genes interact in specific conditions and it allows to detect novel putative pathways associated with cancer
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